The use of monoclonal antibodies (“mAbs”) as therapeutic treatment for a variety of diseases and disorders is rapidly increasing because they have been shown to be safe and efficacious therapeutic agents. Approved therapeutic monoclonal antibodies in the United States include ReoPro® (for patients undergoing high-risk coronary angioplasty to prevent recurrent coronary obstruction), Herceptin (for treatment of metastatic breast cancer), Zenapax (for prevention of acute kidney transplant rejection), Synagis (for the treatment of RSV-induced lower respiratory tract disease), Rituxan (for treatment of B cell non-Hodgkin's lymphoma), Campath (for treatment of B cell chronic lymphocytic leukemia), Humira (for the treatment of rheumatoid arthritis) and Remicade (for the treatment of Crohn's Disease). Many more monoclonal antibodies are in various phases of clinical development for a variety of diseases. Perhaps the most prevalent of these potential therapeutics are monoclonal antibodies that target various forms of cancer.
Monoclonal antibodies are typically generated in non-human mammals, most frequently the mouse, by administration of an “immunogen” (a substance or mixture of substances that elicit an antibody response) and subsequent isolation of B-cells that make antibodies. The B-cells are then immortalized by fusion to another, stable cell type to create a “hybridoma”. A central tenet of modern immunology is that an individual B-cell makes one specific antibody that is defined by its primary amino acid sequence and its underlying gene sequence. In other words, each B-cell is clonal and therefore each immortalized hybridoma will make only one specific antibody. Moreover, the ability of a given antibody to bind to an antigen (a foreign entity to the body) is mediated largely by six peptide loops carried on the two prototypical chains that make up an antibody (termed the “heavy” and light” chains). These peptide loops cause the antibody to bind to specific portions (epitopes) of an antigen. Because the binding of antibodies to their epitopes is that of two complementary surfaces interacting, these six peptide loops are termed complementary determining regions, or CDRs.
The therapeutic utility of murine mAbs has been found to be limited, principally due to the fact that human patients mount their own antibody response to murine proteins, including murine mAbs. This so-called HAMA (human anti-mouse antibody) response results in the eventual neutralization and elimination of murine mAbs, rendering these mAbs therapeutically ineffective after only a few administrations. One solution to this problem involves a process termed “humanization” where, in its simplest terms, the murine CDRs of an antibody are “grafted” onto the framework of a human antibody. Advances in the ability to “humanize” monoclonal antibodies have greatly contributed to the efficacy of these molecules as therapeutics. Humanization prevents or greatly delays the patient developing an immune response against the administered therapeutic monoclonal antibody and extends the half-life of that antibody in circulation.
With this solution, one of the major remaining limitations to deriving therapeutic antibodies to a particular antigen is the ability to obtain a mAb that recognizes a desired epitope. Many therapeutic target antigens are poorly immunogenic. They are incapable of stimulating target-specific monoclonal antibody production. The ability of a mouse or a human to recognize an antigen and mount an antibody response is determined in part by its antibody diversity.
The intrinsic antibody diversity in mice and humans is large, but not infinite. Overall, antibody diversity is defined by five steps.
Step 1—Rearrangement of an inherited set of gene segments (termed V, D, or J gene segments) that make up the variable region of immunoglobulin heavy or light chains. Each individual inherits multiple copies of the V, D, and J gene segments and different combinations of these give rise to different heavy or light chains. This process is known as somatic recombination and is tightly regulated as the B cell develops from a hematopoietic stem cell to a pro-B cell, then a pre-B cell and finally to an “immature” B-cell that expresses a specific IgM molecule on its surface.
Step 2—Different combinations of heavy and light chains give rise to different antigen-binding sites (one prototypical immunoglobulin). In theory, these first two processes could give rise to about 3.5 million different antibody specificities in humans (in “Immunobiology: The Immune System in Health and Disease,” 4th Edition, C. A Janeway, P. Travers, and M. Walport [eds.] Garland Publishing, New York, 1999).
Step 3—Additional diversity is then generated by a process that randomly inserts or deletes nucleotides in the third CDR region where different constituent gene segments join. The result of this “functional diversity” is often (roughly 2 out of 3 times) a disruption of the reading frame and the production of a non-functional protein. All mechanisms for generating diversity described so far occur during the development of B cells in the bone marrow. At this point, the maturation of the B cell has progressed from a stem cell to an “immature” B cell.
Step 4—The antibody repertoire is then edited (reduced) by elimination of immature B cells attempting to produce unstable antibodies or the removal of immature B cells that recognize “self”. The latter point is crucial in order to avoid the immune system attacking the organism's own body. This editing of the repertoire occurs for the immature B cell both in the bone marrow and as the B cells first migrate to secondary lymphoid tissue. After further development to full maturation, a population of naïve B cells exists in the periphery that can engage antigens with low affinity. The repertoire of antigen binding sites represented by this population can be thought of as “shape templates” seeking an initial match against foreign antigens. Any naïve B cell that successfully matches an epitope on an antigen is selectively stimulated to proliferate—a process known as positive selection.
Step 5—A final source of immunoglobulin diversity derives from a process known as somatic hypermutation. This process introduces point mutations into the V regions of immunoglobulin genes upon encounter of a mature B cell with an antigen. This mutation of the immunoglobulin gene occurs at a very high rate and results in expression of altered immunoglobulin molecules on the surface of the B cell. Some of these altered immunoglobulins will bind antigen even better than the original and are preferentially selected to mature into antibody-secreting cells. This latter process is known as affinity maturation.
One of the potential reasons that a foreign antigen is poorly immunogenic is that the immune system of a mouse will not produce antibodies against a self-antigen (an antigen that is normally produced by that mouse). If the antigen representing a desired therapeutic target (e.g. a viral or bacterial protein or fragment thereof or a human or other mammalian protein) shares one or more epitopes with a self-antigen, no monoclonal antibodies will be generated against those epitopes. Many pathogens seek to avoid destruction by the host immune system by “masking” their surfaces with host self-antigens.
Returning to Step 4, above, when an immature B cell encounters a self-antigen, it has four possible fates—each of which insure that it will not mature and enter into the animal's circulation. One fate is cell death by apoptosis. This is also known as clonal deletion and is typical for B cells that interact with multivalent self-antigens, such as multiple copies of an MHC molecule on a cell surface. A second potential fate is called receptor editing. In this scenario, the self-reactive B cell can be rescued by further gene rearrangements in immunoglobulin light chains such that the cell no longer produces an antibody against self-antigens.
Self-reactive B cells that recognize smaller antigens respond differently. These cells tend to be inactivated and enter a state of anergy, or permanent unresponsiveness. Such cells do ultimately end up in circulation, but are short-lived and incapable of producing antibodies against self-antigens. The fourth potential fate is that the B cell remains in a state of immunological “ignorance” of the self-antigen. These cells, which may actually be very weakly reactive with self-antigens, enter the circulation and have been speculated to be the source of autoimmune disease.
Thus, the naïve B-cells entering circulation represent one level of antibody diversity, a level of diversity reduced relative to that specified at the genomic level. The cell surface IgM's clonally expressed on each B-cell have a defined three dimensional shape in their antigen binding site (a shape template) that can potentially interact with a complementary shape on a foreign antigen. The ensemble of all B-cells that have exited the bone marrow thus define a repertoire of “shape templates” that make the initial encounter with foreign antigens.
When a foreign antigen is encountered, those naïve B-cells whose surface IgM is complementary with some part of the antigen surface (an epitope) receive intracellular signals to proliferate. This positive selection of a limited subset of circulating B-cells insures adequate numbers of reactive B-cells are produced to overwhelm the foreign agent. Further antibody diversity then ensues (in tandem with selective proliferation) through a process known as somatic hypermutation. In this process, random nucleic acid substitutions are made in the region coding for the antigen-binding site and further positive selection is made for those new antibodies that bind with higher affinity to the antigen (better and/or more extensive shape complementation between the B-cell's surface immunoglobulin and the antigen). Somatic hypermutation has been estimated to expand the ultimate scope of antibody diversity 10 to 100-fold or more. Thus, any process that can expand the repertoire of “shape templates” will result in a disproportionate expansion (through somatic hypermutation) in the ultimate scope of antibody diversity in a mouse or human that can respond to a foreign antigen.
Post-translational modification of proteins, such as glycosylation, sulfation, acetylation and proteolytic processing, and modification of lipids, such as glycosylation, result in antigenic epitopes that can be recognized by monoclonal antibodies. Because many of these same modifications are present on self-antigens, such epitopes do not typically stimulate antibody production in a normal, wild-type mouse. However, mice that are defective in one or more enzymes involved in such modifications (knockout mice) will produce native proteins and lipids with altered modifications (truncated, altered or complete lack of the modification). These knockout mice are capable of generating an antibody response to corresponding wild-type modified proteins and lipids because their immune system does not see such molecules as self-antigens. Such antibodies may be generated by the wild-type protein or lipid or, in some cases, by foreign antigens that share epitopes with the wild-type protein or lipid.
Recent experiments have confirmed this in an experiment designed to generate antibodies to naturally occurring murine brain gangliosides (glycolipids) in order to study the expression and function of these molecules, as well as ganglioside-related pathologies (R. L. Schnaar et al., Anal Biochem., 302, pp. 276-284 (2002)). This group demonstrated that immunization of mice defective in a key enzyme in major brain ganglioside biosynthesis (UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) with wild-type gangliosides resulted in the production monoclonal antibodies that recognized those wild-type gangliosides. Similar immunization of wild-type mice did not result in the production of any monoclonal antibodies that recognized the wild-type gangliosides. Similar results were also obtained by another group using mice knocked out for the same gene (M. P. T. Lunn et al., J. Neurochem., 75, pp. 404-12 (2000).
Another group used lipopolysaccharides from the bacteria C. jejuni were used as an antigen to raise antibodies in the same knockout mouse. Those mice produced antibodies that cross-reacted with those wild-type mouse gangliosides which were lacking in the knockout mouse (T. Bowes et al., Infect. Immun., 70, pp. 5008-18 (2002). The same antigens in wild-type mice did not produce a significant antibody response against either the antigen itself or any mouse gangliosides.
In addition, knockout mice with targeted inactivation of post-translational modifications will possess naïve B cells with an increased repertoire of “shape templates” that initially engage antigens. If these additional “shape templates” interact with foreign antigens, whatever the composition of the epitope, the process of somatic hypermutation will greatly amplify the diversity of antibodies made in response to that antigen as compared to the wild type mouse and these additional antibodies will be unique to the knockout mouse.
Despite the advances that have been made in generating therapeutically effective monoclonal antibodies, there still exists a need for generating a greater diversity of monoclonal antibodies against therapeutically important targets (C. A. K. Borrebaeck and M. Olin (2002) Nature Biotechnology 20:1189-1190). Because human disease is often marked by the utilization of some aspect of “self” to avoid host immune surveillance or as a consequence of the pathological process, there is a need for methods of generating monoclonal antibodies against targets that share epitopes with self antigens and which cannot be produced using wild-type mice.